Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Summary

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Abstract

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.

Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [³H]2-deoxy-D-glucose and [¹⁴C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Introduction

Skeletal muscle possesses the ability to extract large quantities of glucose from the extracellular space in response to insulin and exercise. This helps to maintain whole-body glucose homeostasis and secures glucose supply during times of high energy demand. Since intact regulation of skeletal muscle glucose uptake has been shown to be important for overall health and physical performance^1,2, measurements of muscle glucose uptake during various conditions have received much attention. In humans and animals, the hyperinsulinemic-euglycemic clamp has been used as the gold standard technique to assess insulin sensitivity in vivo^3,4. In contrast to findings obtained from an oral glucose tolerance test, the hyperinsulinemic-euglycemic clamp technique does not require intact gastrointestinal function or insulin secretion from the pancreas and thus permits insulin responses to be compared between subjects who exhibit variations in gastro-intestinal and/or pancreatic function. Measurements of muscle glucose uptake in vivo during exercise in humans have been performed frequently since the 1960s⁵. First by the use of arteriovenous balance techniques⁶ and later by the use of positron emission tomography (PET) imaging in combination with a positron emitting glucose analogue e.g. ¹⁸F-Fluoro-deoxy-glucose⁷. In rodents, exercise-stimulated muscle glucose uptake in vivo is typically performed by the use of radioactive or stable isotope-labeled glucose analogues^8,9,10.

A complementary method to measurements of muscle glucose uptake in vivo, is to isolate and incubate small muscles from rodents and subsequently measure glucose uptake using radioactive or stable isotope-labeled glucose analogues^11,12,13. This method allows accurate and reliable quantification of glucose uptake rates in mature skeletal muscle and can be performed in the presence of various insulin concentrations and during contraction elicited by electrical stimulation. More importantly, measurements of glucose uptake in isolated and incubated skeletal muscle are of relevance when investigating the muscle metabolic phenotype of mice that have undergone various interventions (e.g. nutrition, physical activity, infection, therapeutics). The isolated skeletal muscle model is also a suitable tool for pharmacological compound testing that may affect glucose uptake per se and/or modify insulin sensitivity^12,14. In this way, the efficacy of compounds designed to regulate muscle glucose metabolism can be tested and evaluated in a highly controlled milieu before subsequent in vivo testing in pre-clinical animal models.

Under some conditions, metabolic viability may pose a challenge in the isolated and incubated skeletal muscle model system. Indeed, the lack of a circulatory system in the incubated muscles entails that delivery of substrates (e.g. oxygen and nutrients) fully depends on simple diffusion between the muscle fibers and the surrounding environment. In regards to this, it is of importance that the incubated muscles are small and thin and thus, represent less of a barrier for oxygen diffusion during incubation¹⁵. Especially during prolonged incubations for several hours, hypoxic states may develop due to insufficient oxygen supply resulting in muscle energy depletion¹⁵. Although various markers of metabolic viability in incubated rat muscle have been reported previously alongside the identification of important variables that help to maintain rat muscle viability¹⁵, a comprehensive evaluation of metabolic viability in small incubated mouse muscles is still warranted. Hence, at present, glycogen content has mainly been used as a marker of metabolic viability in incubated mouse skeletal muscle^16,17.

Here we describe a detailed protocol to measure basal, insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle from mice using radiolabeled [³H]2-deoxy-D-glucose and [¹⁴C]mannitol as an extracellular marker. In the present study, glucose uptake was measured during a 10-minute period and the method is presented with the use of submaximally and maximally effective insulin concentrations as well as a single contraction protocol. However, the protocols described herein can easily be modified with regards to incubation time, insulin-dosage, and electrical stimulation protocol. Furthermore, we provide a thorough characterization of various markers of metabolic viability in incubated soleus and EDL mouse muscle. The results indicate that glucose supplementation to the incubation buffer is essential to preserve metabolic viability of muscle incubated for 1 hour.

Protocol

Procedures involving research animals should be performed in accordance with relevant guidelines and local legislation. All animal experiments used for this study complied with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes and were approved by the Danish Animal Experiments Inspectorate. 

1. Preparation of the experimental apparatus and suture loops

NOTE: For this study, use an integrated muscle strip myograph system with customized incubation hooks to incubate isolated mouse skeletal muscles (Figure 1). This system allows muscle to bathe in a physiological solution with continuous oxygenation (95% O₂ and 5% CO₂) and at constant temperature. The muscle tissue bath is coupled to a force transducer for the measurement of muscle force production during contraction. To elicit and record myo-mechanical responses during contraction, employ an electrical pulse stimulator and a data collection program, respectively. Stimulate the incubated muscles to contract by platinum electrodes positioned centrally and on both sides of the muscle.

1. Turn on the myograph system and warm chambers to 30 °C. Open data collection software compatible with the myograph system and calibrate force transducers to ensure comparability between datasets.
2. Start by cutting ~16 cm strands of non-absorbable surgical nylon suture. Use forceps to create a loop of approximately 0.4 cm in diameter from a single strand. Repeat this until enough loops have been produced. Each muscle needs two loops - one for the proximal and one for the distal tendon.

2. Preparation of solutions and incubation media

1. Preparation of basal incubation media
   1. Prepare the following stock solutions: 2.5 M sodium chloride (NaCl, 250 mL), 0.5 M sodium bicarbonate (NaHCO₃, 250 mL), 0.5 M potassium chloride (KCl, 50 mL), 0.25 M calcium chloride (CaCl₂, 50 mL), 0.25 M potassium dihydrogen phosphate (KH₂PO₄, 50 mL), 0.25 M magnesium sulfate (MgSO₄, 50 mL), 110 mM sodium pyruvate (Na-Pyruvate, 100 mL), 500 mM D-mannitol (100 mL), 1 M 2-deoxy-D-glucose (4 mL), 15% solution of bovine serum albumin (BSA) dialyzed against Krebs-Ringer-Henseleit (KRH) buffer (described below in step 4) (100 mL).
      ​NOTE: Two solutions are required to measure resting and contraction-stimulated glucose uptake. Furthermore, each single insulin concentration used to assess insulin-stimulated glucose uptake requires two solutions. Thus, in total six different solutions are needed to measure basal, submaximal insulin-, maximal insulin-, and contraction-stimulated glucose uptake in isolated mouse skeletal muscle. In the following, 'basal incubation media' refers to media without insulin or radioactive tracers. 'Incubation media' refers to media containing insulin. 'Glucose uptake incubation media' refers to media containing 2-deoxy-D-glucose and radioactive tracers in addition to insulin at a concentration identical to that used in the 'incubation media'.
   2. Prepare a KRH buffer by supplementing ultrapure water (ddH₂O) with NaCl (117 mM), NaHCO₃ (24.6 mM), KCl (4.7 mM), CaCl₂ (2.5 mM), KH₂PO₄ (1.2 mM), and MgSO₄ (1.2 mM). Subsequently, gas the KRH buffer with 95% O₂ and 5% CO₂ for at least 10 min. The desired pH of the KRH buffer should be between 7.35-7.45 at 30 °C. If pH adjustment is performed at room temperature the pH of the KRH buffer should be between 7.25-7.35.
   3. Add BSA (0.1%), Na-Pyruvate (2 mM), and D-mannitol (8 mM) to the gassed and pH-adjusted KRH buffer to complete the basal incubation media. Store basal incubation media in a sealed container to minimize degasification of O₂ and CO₂ and place media at 30 °C.
      NOTE: Typically, the osmolarity of the KRH supplements (i.e. Na-Pyruvate, D-Mannitol, and D-glucose) is kept constant across an entire experiment to avoid shrinkage or expansion of the muscle cells. The protocol described herein uses an osmolarity of 10 mM for the KRH supplements. If a glucose-containing buffer is needed, replace KRH supplements to accommodate the needs, e.g. 5 mM D-glucose and 5 mM D-mannitol.
   4. To avoid possible BSA-associated contaminants in the incubation buffer, dialyze BSA against KRH.
      1. To make a 15% BSA stock solution dialyzed against KRH buffer, start by dissolving 300 g of analytical grade fat-free BSA in 900 mL of KRH buffer. Next, boil the dialysis tube in redistilled water until the tubing is soft.
      2. Fill the tubing with the BSA-KRH solution and secure the tubing ends. Place the tubing with BSA-KRH in 5 L of KRH buffer and leave it overnight at 4 °C. The following day replace the KRH buffer and leave the tubing with BSA-KRH in KRH buffer overnight at 4 °C.
      3. Lastly, collect the BSA-KRH solution from the tubing and add KRH buffer to a final volume of 2 L (i.e., 15% BSA-KRH stock solution). Divide the 15% BSA-KRH stock solution into aliquots and store in freezer at ~-20 °C.
2. Preparation of incubation media containing insulin
   1. For the incubation media containing a submaximally effective insulin concentration, add 1 µL of a 100 mU/mL insulin stock solution per mL of basal incubation media (100 µU/mL insulin).
   2. For the incubation media containing a maximally effective insulin concentration, add 1 µL of a 10 U/mL insulin stock solution per mL of basal incubation media (10 mU/mL insulin).
3. Preparation of glucose uptake incubation media
   ​​CAUTION: Handling of radioactive material is only allowed in a restricted and controlled area by authorized personnel and some universities, research institutions and companies may require the acquisition of a "Radioactivity Use Permit". Material and waste must be handled according to appropriate local procedures, guidelines, and legislation.
   1. Follow the same procedure as described in section 2.1.2.
   2. Add BSA (0.1%), Na-Pyruvate (2 mM), D-mannitol (7 mM), and 2-deoxy-D-glucose (1 mM) to the gassed and pH-adjusted KRH buffer.
   3. Add [³H]2-deoxy-D-glucose (0.028 MBq/mL) and [¹⁴C]mannitol (0.0083 MBq/mL) to the supplemented KRH buffer to complete the glucose uptake incubation media. Store at 30 °C. If [³H]2-deoxy-D-glucose and [¹⁴C]mannitol are dissolved in ethanol remove the ethanol by N₂ mediated evaporation before use.
   4. For the glucose uptake incubation media containing a submaximally effective insulin concentration, add 1 µL of a 100 mU/mL insulin stock solution per mL of glucose uptake incubation media (100 µU/mL insulin).
   5. For the glucose uptake incubation media containing a maximally effective insulin concentration, add 1 µL of a 10 U/mL insulin stock solution per mL of glucose uptake incubation media (10 mU/mL insulin).

3. Animals and dissection of the mouse soleus and EDL muscle for incubation

NOTE: Procedures involving research animals should be performed in accordance with relevant guidelines and local legislation. The described procedure can be used with in-house bred or commercially available male and female mice of various strains and genetic backgrounds. The following procedure is provided for fed female C57Bl/6J mice. On average, mice were 19 weeks old and weighed 25 g. The mice were maintained on a 12:12 h light-dark cycle with free access to standard rodent chow and water. Animal experiments were initiated at ~ 9:00 AM local time and all animals were sacrificed within a period of 2 h.

1. Add 4 mL of pre-warmed (30°C) basal incubation media to each incubation chamber and make sure the basal incubation media is continuously oxygenated with 95% O₂ and 5% CO₂.
2. Anaesthetize mice with an intraperitoneal injection of pentobarbital (10 mg/100 g body weight) or other available anaesthesia (e.g. tribromoethanol).
   ​NOTE: Be aware that in some countries a licence to handle pentobarbital and other anaesthetic drugs may be required. Before muscle dissection can be initiated, anaesthesia of each animal must be properly induced. To ensure this, tail and leg reflexes are tested. For optimal results dissection should be well practiced to avoid damaging the muscles during removal.
3. Place anaesthetized mice prone on a dissection tray (e.g. styrofoam lid) and pin down front and hind paws, as necessary, using a needle.
4. Remove the skin from the lower leg and make sure that both the Achilles tendon and knee joint are visible.
   1. For the dissection of soleus muscle, start by attaching a single suture loop to the Achilles tendon. Secure a pean forceps to the Achilles tendon distally of the suture loop and cut to release the soleus and gastrocnemius muscles from the paw. Carefully slide the pean forceps across the mouse thereby exposing the soleus muscle.
   2. Pin down the pean forceps and place a second suture loop around the proximal tendon of the soleus muscle. Next, cut the proximal tendon and dissect soleus (including the two attached suture loops) free of gastrocnemius muscle. Quickly place the soleus muscle in the incubation chamber by attaching each suture loop to the respective hooks.
5. Remove the fascia covering the m. tibialis anterior (TA) using forceps. If done correctly, distal tendons of the TA and EDL muscles should be clear white and visible; and separated from each other.
6. Cut the distal tendon of the TA muscle and dissect out the muscle for later analyses (e.g. genotyping). Using forceps, gently liberate the EDL muscle from the surrounding tissues but leave the muscle intact and do not cut the tendons. Place one suture loop around the distal tendon and a second suture loop around the proximal tendon of EDL.
7. Next, cut the tendons releasing the EDL muscle with two attached suture loops and quickly place the muscle in the incubation chamber by attaching each suture loop to the respective hooks. In order to not lose tension during incubation and especially during electrically-induced contraction of the soleus and EDL muscles, it is of great importance to fix the suture loops around the tendons with tight knots.
8. Lastly, euthanize the animal by e.g. cervical dislocation.
9. When the muscles have been dissected and placed in incubation chambers, adjust the resting tension of each muscle to ~5 mN and pre-incubate the muscles for at least 10 min before initiating the experimental protocol.

4. Insulin-stimulated glucose uptake in isolated mouse skeletal muscle

1. Following step 3.9 replace the basal incubation media with incubation media containing no insulin (basal incubation media), a submaximally effective insulin concentration or a maximally effective insulin concentration and leave in the incubation chambers for 20 min. Space each incubation chamber by 1 min, thereby making time for the subsequent harvest of muscles.
2. At the end of the 20 min stimulation period, replace the incubation media with the glucose uptake incubation media containing an identical concentration of insulin and leave in the incubation chambers for 10 min, again with 1 min spacing between each incubation chamber.
3. After 10 min of incubation in the glucose uptake incubation media gently remove muscles from the incubation chambers and wash them in ice-cold basal incubation media. Subsequently, quickly dry the muscles on filter paper before the suture loops are removed and muscles are frozen in liquid nitrogen. It is imperative that the incubated muscles are harvested quickly if one also wishes to investigate various intracellular metabolites and protein signaling in addition to glucose uptake.
4. Collect 100 µL of the glucose uptake incubation media from each incubation chamber and store it at -20 °C. The amount of radioactivity in these samples will be included in the calculation of muscle glucose uptake.

5. Contraction-stimulated glucose uptake in isolated mouse skeletal muscle

NOTE: To induce contraction of isolated mouse skeletal muscle use the following protocol: 1 train/15 s, each train 1 s long consisting of 0.2 ms pulses delivered at 100 Hz. However, other similar protocols eliciting contraction of isolated mouse skeletal muscle will likely work as well. Importantly, the voltage should be adjusted to generate maximal force development of the incubated muscle, which is dependent on the experimental setup. If this is not ensured, you may risk that not all fibers of the muscle are contracting. In turn, this may induce bias in the dataset.

1. Following step 3.9 place the platinum electrodes centrally and on both sides of the muscles. Initiate contraction of the muscles immediately after replacing the basal incubation media with the glucose uptake incubation media. If possible, space each incubation chamber by 1 min, thereby making time for the subsequent harvest of muscles. Remember to record force production from each incubated muscle.
2. After 10 min of contraction in the glucose uptake incubation media, remove the platinum electrodes, gently collect the muscles from the incubation chambers and wash them in ice-cold basal incubation media. Subsequently, quickly dry the muscles on filter paper before the suture loops are removed and muscles frozen in liquid nitrogen. The entire muscle harvest procedure should be performed as fast as possible.
3. Collect 100 µL of the glucose uptake incubation media from each incubation chamber and store it at -20 °C. The amount of radioactivity in these samples will be included in the calculation of muscle glucose uptake.

6. Skeletal muscle homogenization and processing

NOTE: The procedure given below for muscle homogenization makes it possible to determine both glucose uptake and myocellular signaling by western blotting in the same set of muscle samples.

1. Homogenize each muscle in 400 µL of ice-cold buffer with pH 7.5 containing 10% glycerol, 20 mM sodium-pyrophosphate, 1% IGEPAL CA-630 (NP-40), 2 mM phenylmethylsulfonylfluoride (dissolved in isopropanol), 150 mM NaCl, 50 mM HEPES, 20 mM β-glycerophosphate, 10 mM sodium fluoride (NaF), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM glycoletherdiaminetetraacetic acid (EGTA), 10 µg/ml aprotinin, 10 µg/mL leupeptin, 3 mM benzamidine, and 2mM sodium-orthovanadate using steel beads and a tissuelyser (2 x 45 s at 30 Hz). Rotate all homogenates end-over-end for 1 h at 4 °C after which they are centrifuged at 16,000 x g for 20 min at 4 °C. Collect the lysate (supernatant) which is used to determine muscle glucose uptake.

7. Determination of radiolabeled 2-deoxyglucose and mannitol

1. Add 150 µL of each muscle lysate and 25 µL of the glucose uptake incubation media from each incubation chamber to separate liquid scintillation counting vials containing 2 mL of liquid scintillation fluid. Moreover, prepare two blind control vials only containing 3 mL of liquid scintillation fluid. Close all the vials and mix thoroughly by vortexing each vial for ~5 s.
2. Place the vials in a liquid scintillation counter and measure radioactivity of [³H]2-deoxy-D-glucose and [¹⁴C]mannitol according to the manufacturer's guidelines. Record DPM (disintegrations per minute) for each liquid scintillation vial.

8. Calculation of muscle glucose uptake rates

1. Use the lysate from step 6.1 to measure the total protein concentration in each muscle sample using standard protein quantification methods (e.g., Bicinchoninic acid or Bradford assays). Calculate the amount of protein (mg) added to each scintillation vial.
   NOTE: The rate of glucose uptake for each muscle sample is calculated by subtracting the amount of [³H]2-deoxy-D-glucose located in the extracellular space from the total amount of [³H]2-deoxy-D-glucose in the muscle sample using [¹⁴C]mannitol as an extracellular marker. It is assumed that [³H]2-deoxy-D-glucose and [¹⁴C]mannitol exhibit similar diffusion properties within the muscle tissue during incubation. Perform the following calculations:
2. Start by subtracting the [³H] and [¹⁴C] DPM of the blind control samples from all muscle and media samples.
3. Determine the muscle extracellular space in µL (µL-ECS):
   [¹⁴C]DPM_muscle / ([¹⁴C]DPM_media / M_vol)
4. Calculate the amount of [³H]DPM in the muscle extracellular space ([³H]DPM_ECS):
   µL-ECS × ([³H]DPM_media / M_vol)
5. Calculate the amount of [³H]DPM in the muscle intracellular space ([³H]DPM_ICS):
   [³H]DPM_muscle− [³H]DPM_ECS
6. Calculate the muscle glucose uptake rate (µmol / g protein / hour):
   [³H]DPM_ICS / ([³H]DPM_media / M_vol) / [2-deoxy-D-glucose])) / mg protein) / T_h
   NOTE: For all the equations above,
   [¹⁴C]DPM_muscle is the amount of [¹⁴C]mannitol radioactivity in a muscle sample;
   [¹⁴C]DPM_media is the amount of [¹⁴C]mannitol radioactivity in a media sample;
   [³H]DPM_muscle is the amount of [³H]2-deoxy-D-glucose radioactivity in a muscle sample;
   [³H]DPM_mediais the amount of [³H]2-deoxy-D-glucose radioactivity in a media sample;
   [³H]DPM_ECS is the amount of [³H]2-deoxy-D-glucose radioactivity in the muscle extracellular space;
   [³H]DPM_ICS is the amount of [³H]2-deoxy-D-glucose radioactivity in the muscle intracellular space;
   µL-ECS is the muscle extracellular space in µL;
   M_vol is the volume (µL) of incubation media used for scintillation counting (e.g. '25' as mentioned above);
   T_h is the time factor used to calculate uptake rates per hour (i.e., '1/6' when incubating muscles with glucose uptake media for 10 min)
7. Take into consideration this example calculation. The [³H] and [¹⁴C] DPM of the blind control samples (17 and 6, respectively) have been subtracted from the DPM values mentioned below.
   [¹⁴C]DPM_muscle: 343
   [¹⁴C]DPM_media: 11846
   [³H]DPM_muscle: 4467
   [³H]DPM_media: 39814
   M_vol: 25
   mg protein: 0.396 (in 150 µL muscle protein lysate)
   [2-deoxy-D-glucose]: 1 (mM)
   T_h: 1/6 (h)
   µL-ECS = 343 DPM / (11846 DPM / 25 µL) = 0.724 µL
   [³H]DPM_ECS: 0.724 µL × (39814 DPM / 25 µL) = 1153 DPM
   [³H]DPM_ICS: 4467 DPM - 1153 DPM = 3314 DPM
   Glucose uptake: ((3314 DPM / (39814 DPM / 25 µL) / 1 mmol/L) / 0.396 mg protein) / (1/6 hour) = 31.53 µmol / g protein / hour

9. SDS-PAGE and western blot analyses

1. Prepare soleus and EDL muscle lysates in Laemmli buffer and heat for 5 min at 96 °C.
2. Separate equal amounts of muscle protein by SDS-PAGE on self-cast gels and transfer the proteins to polyvinylidene fluoride membranes by semidry blotting.
3. Subsequently, incubate membranes in Tris-buffered saline containing 0.05% Tween 20 and 2% skim milk and probe membranes with relevant primary and secondary antibodies.
4. Detect proteins with chemiluminescence and visualize them by a digital imaging system.

10. Muscle glycogen, nucleotides, lactate, creatine, and phosphocreatine

1. Use perchloric acid to extract EDL and soleus muscle samples.
2. Subsequently, neutralize samples and analyze them for lactate, creatine, and phosphocreatine as previously described¹⁸.
3. Analyze nucleotide content in EDL and soleus muscle by reverse-phase HPLC following extraction in perchloric acid.
4. Determine muscle glycogen content in whole muscle homogenate as glycosyl units after acid hydrolysis by a fluorometric method as previously described¹⁸.

11. Statistics

1. Perform statistical analyses with statistical analyses software.
2. Use a two-way analysis of variance (ANOVA) test to assess statistical differences between values presented in Table 1.
3. Use unpaired Student t tests to assess statistical differences in glucose uptake between EDL and soleus within each group presented in Figure 2. Present data as means ± standard error of the mean (SEM). P < 0.05 is considered statistically significant.

Representative Results

As shown in Figure 2 the basal glucose uptake rates were similar between isolated soleus and EDL muscle from female mice. This has also been reported several times before^12,13,19,20. Glucose uptake increased by ~0.8 and ~0.6 fold reaching 12 and 9 µmol/g protein/h in soleus and EDL muscle, respectively, in response to a submaximally effective insulin concentration (100 µU/mL). This increase was even higher (~4 and ~2 fold reaching 33 and 19 µmol/g protein/h in soleus and EDL muscle, respectively) when muscles were stimulated with a maximally effective insulin concentration (10 mU/mL). Moreover, both submaximal and maximal insulin-stimulated glucose uptake were significantly higher in soleus muscle indicating that soleus muscle exhibits enhanced insulin sensitivity and responsiveness compared to EDL muscle. This may be related to the higher expression of the glucose transporter 4 (GLUT4) as well as the insulin signaling transducer protein kinase B (Akt) in soleus muscle compared to EDL muscle^{10,21,22,23,24}.

Contraction-induced glucose uptake was significantly higher in EDL muscle compared to soleus muscle (Figure 2) as also previously reported^13,19. Thus, glucose uptake increased by ~2 and ~2.5 fold reaching 14 and 22 µmol/g protein/h in soleus and EDL muscle, respectively, in response to electrically-induced contractions. Figure 3 shows maximal muscle force production in soleus and EDL muscle during the 10 min stimulation period. As seen and previously reported¹⁹, the EDL muscle generates more force (225 mN in EDL vs. 150 mN in soleus) during the initial part of the stimulation period. On the other hand, the EDL muscle exhibits a faster decline in force production compared to the soleus muscle later on in the stimulation period. These findings are likely due to the difference in fiber type distribution between the soleus (type 1 > type 2) and EDL (type 2 > type 1) muscle²⁵ as type 2 fibers generate more force but fatigue faster compared to type 1 fibers^26,27.

To evaluate the effect of insulin and contraction on intracellular signaling in isolated soleus and EDL muscle, phosphorylation of Akt Thr308, TBC1 domain family member 4 (TBC1D4) Ser588, AMPKα Thr172 and acetyl-CoA carboxylase (ACC) Ser212 was performed by the western blotting technique (Figure 4). As expected, the submaximally and maximally effective insulin concentration induced an increase in the phosphorylation of Akt Thr308 and TBC1D4 Ser588 while contraction induced an increase in the phosphorylation of AMPKα Thr172 and ACC Ser212. Neither insulin nor contraction led to a change in the total protein content of Akt2, TBC1D4, AMPKα2 and ACC in soleus and EDL muscle (Figure 4).

Examining various markers of metabolic viability of incubated soleus and EDL muscle, we observed an overall reduction in the levels of adenosine nucleotides (ATP, ADP, AMP) (~15-25%) as well as creatine (~10-35%) regardless of whether the muscles were incubated in the presence of pyruvate or glucose compared to non-incubated muscles (Table 1). On the other hand, the drop in glycogen levels observed in soleus and EDL muscles incubated with pyruvate was prevented if the muscles were incubated with glucose. Interestingly though, we observed that the inosine monophosphate (IMP) levels increased several fold but only in incubated soleus muscle. IMP levels typically increase in muscle during severe metabolic stress as the muscle attempts to prevent AMP accumulation by converting AMP into IMP in order to maintain the ATP/ADP ratio²⁸. This indicates that the soleus muscle is somewhat more metabolically stressed compared to the EDL muscle during incubation. This notion is also supported by findings of elevated AMPKα Thr172 and ACC Ser212 phosphorylation in the soleus muscle incubated with pyruvate (Figure 5). Importantly, the observed increase in IMP levels as well as AMPKα Thr172 and ACC Ser212 phosphorylation decrease when the soleus muscle is incubated with glucose. Hence, it seems advantageous to incubate isolated skeletal muscle in a glucose-containing buffer to minimize fluctuations in adenosine nucleotides and prevent a drop in muscle glycogen when muscles are incubated for a prolonged period of time. With regards to 2-deoxyglucose uptake, we have evidence to suggest that incubating muscles for 6 to 8 h in the presence of pyruvate will increase basal/resting glucose uptake rates. Incubating muscles in a media containing glucose seems to prevent such increase in glucose uptake (unpublished data).

Figure 1: Incubation system. (A) Myograph system with four single incubation chambers. (B) Customized incubation hooks. Please click here to view a larger version of this figure.

Figure 2: Glucose uptake in isolated mature skeletal muscle from mice. 2-deoxyglucose uptake was determined in isolated soleus (black bars) and EDL (gray bars) muscles in response to a submaximally effective insulin concentration (100 µU/mL), a maximally effective insulin concentration (10 mU/mL), and electrically-induced contractions (0.2 ms pulse, 100 Hz, 1 s/15s, 30 V, 10 min). Data were analyzed by Students t test within each group. ###p<0.001, ##p<0.01 vs. soleus muscle. Values are means ± SEM. n = 4-6 per group. h, hour. Please click here to view a larger version of this figure.

Figure 3: Muscle force curves in response to electrically induced contractions. Peak force production during electrical stimulation was calculated for the soleus (black dots) and EDL (gray dots) muscle. Each single value corresponds to an average of the last 500 ms of each 1-s stimulation period. Values are means ± SEM. n = 5-6 per group. s, second. mN, milli-Newton. Please click here to view a larger version of this figure.

Figure 4: Representative western blots of Akt Thr308, TBC1D4 Ser588, AMPKα Thr172 and ACC Ser212 phosphorylation as well as Akt2, TBC1D4, AMPKα2 and ACC protein. Western blot analyses were performed on the mouse soleus and EDL muscle samples described in Figure 2. B, basal. S, submaximally effective insulin. M, maximally effective insulin. C, contraction. Please click here to view a larger version of this figure.

Figure 5: Representative western blots of AMPKα Thr172 and ACC Ser212 phosphorylation as well as AMPKα2 and ACC protein. Western blot analyses were performed on the mouse soleus and EDL muscle samples described in Table 1. Please click here to view a larger version of this figure.

	non-incubated		Incubated 1h with pyruvate		Incubated 1h with glucose		Main effects	Interaction
	Soleus	EDL	Soleus	EDL	Soleus	EDL
Lactate	1.00 ± 0.01	1.00 ± 0.02	0.96 ± 0.03	0.98 ± 0.02	1.05 ± 0.01	0.99 ± 0.01	-	-
Cr	1.00 ± 0.04	1.00 ± 0.06	0.64 ± 0.07 ###	0.76 ± 0.05 ###	0.76 ± 0.07 ##	0.88 ± 0.09 ##	p < 0.001	-
PCr	1.00 ± 0.19	1.00 ± 0.06	0.80 ± 0.12	1.13 ± 0.14	0.65 ± 0.31	0.75 ± 0.08	-	-
PCr/(Cr + PCr)	1.00 ± 0.20	1.00 ± 0.07	1.17 ± 0.11	1.25 ± 0.12	0.78 ± 0.36	0.92 ± 0.11	-	-
ATP	1.00 ± 0.03	1.00 ± 0.02	0.72 ± 0.03 ###,§§§	0.99 ± 0.03 *	0.81 ± 0.06 ###	0.85 ± 0.04 ##	-	p < 0.001
ADP	1.00 ± 0.04	1.00 ± 0.04	0.75 ± 0.05 ###	0.92 ± 0.03 ###	0.84 ± 0.04 ##	0.86 ± 0.03 ##	p < 0.001	-
AMP	1.00 ± 0.11	1.00 ± 0.12	0.85 ± 0.15	0.79 ± 0.13	0.84 ± 0.18	0.75 ± 0.13	-	-
IMP	1.00 ± 0.17	1.00 ± 0.30	4.43 ± 0.67 ###,§§§	0.72 ± 0.29	3.33 ± 1.25 ##,§	1.08 ± 0.01	-	p < 0.001
AMP/ATP ratio	1.00 ± 0.12	1.00 ± 0.13	1.18 ± 0.22	0.81 ± 0.16	1.06 ± 0.23	0.90 ± 0.19	-	-
Glycogen	1.00 ± 0.08	1.00 ± 0.12	0.74 ± 0.07 (#),*	0.80 ± 0.12 (#),*	1.12 ± 0.10	1.10 ± 0.03	p = 0.035	-
pAMPK Thr172 / AMPKα2	1.00 ± 0.10	1.00 ± 0.12	3.26 ± 0.58 ###,***,§§§	1.55 ± 0.27	1.68 ± 0.19 #	1.36 ± 0.19	-	p = 0.002
pACC Ser212 / ACC	1.00 ± 0.18	1.00 ± 0.12	2.22 ± 0.58 ###,**,§§	0.96 ± 0.21	0.99 ± 0.15	0.83 ± 0.09	-	p = 0.030

Table 1: Comparison of metabolic viability of mouse soleus and EDL muscles incubated for 1 hour in the presence of 2 mM pyruvate or 5 mM glucose. Non-incubated muscles were dissected from anaesthetized and fed animals before being frozen in liquid nitrogen. Separate muscles were incubated for 1 h in KRH buffer supplemented with BSA (0.1%), Na-pyruvate (2 mM) and D-mannitol (8 mM) while others were incubated for 1 h in KRH buffer supplemented with BSA (0.1%), D-glucose (5 mM) and D-mannitol (5 mM) before frozen in liquid nitrogen. Data were converted into relative units to highlight observed changes in various markers of metabolic viability. Absolute values from non-incubated mouse soleus and EDL muscles are given below. Lactate (mmol/kg w.w): 137.53_soleus; 139.05_EDL. Cr (mmol/kg w.w): 9.35_soleus; 8.98_EDL. PCr (mmol/kg w.w): 1.50_soleus; 5.20_EDL. PCr/(PCr+Cr): 13.98_soleus; 37.30_EDL. ATP (mmol/kg w.w): 3.07_soleus; 4.24_EDL. ADP (mmol/kg w.w): 0.60_soleus; 0.51_EDL. AMP (mmol/kg w.w): 0.18_soleus; 0.08_EDL. IMP (mmol/kg w.w): 0.07_soleus; 0.14_EDL. AMP/ATP ratio: 0.06_soleus; 0.02_EDL. Glycogen (pmol/µg protein): 77.01_soleus; 67.56_EDL. Data were analyzed by a two-way ANOVA within each group. ###p<0.001, ##p<0.01, and #p<0.05 vs. non-incubated. ***p<0.001, **p<0.01, and *p<0.05 vs incubated 1 h with glucose. §§§p<0.001, §§p<0.01, and §p<0.05 vs EDL. Values are means ± SEM. n = 12 in non-incubated group, n = 4-6 in incubated groups. Cr, Creatine; PCr, Phosphocreatine; w.w, wet weight; h, hour.

Discussion

Intact regulation of glucose uptake in skeletal muscle is important for preserving overall health¹. Thus, investigation of muscle glucose uptake often serves as a primary readout when evaluating various health-altering interventions. Here we describe an ex vivo method for measuring glucose uptake in isolated and incubated soleus and EDL muscle from mice in response to insulin and electrically-induced contractions. The method is quick and reliable and allows a precise control of the surrounding milieu of the incubated muscle that permits accurate investigations of muscle glucose uptake rates isolated from the potentially confounding influence of hormones and substrates that can be found in the blood. The method has been used for several years in many studies and is widely adopted by the muscle research community.

The ex vivo incubation model has generally been considered a method to assess glucose transport capacity rather than glucose uptake in skeletal muscle. Glucose transport capacity in incubated muscle can be determined by measuring accumulated D-glucose over a period of time. However, this poses a problem as D-glucose is quickly metabolized in the muscle cell following uptake. To circumvent this problem, the glucose analogue 3-O-Methyl-D-glucose (3-MG) has been widely used to assess glucose transport capacity, as 3-MG is not further metabolized inside the cell after being transported across the cell surface membrane. Thus, the initial rate of intracellular accumulated 3-MG serves as an index of cellular glucose transport capacity per se because it is not affected by other steps in the glucose metabolic pathways. However, the use of 3-MG may constitute a problem as 3-MG will accumulate thereby reducing the transmembrane gradient for 3-MG and subsequently reduce further uptake. Thus, to obtain a measure of the membrane transport capacity, the initial rate of 3-MG uptake must be estimated. In particular when transport capacity is high this may pose a problem due to efflux of 3-MG from the muscle^29,30. The potential problem with 3-MG efflux can be avoided using 2-deoxy-D-glucose (2-DG). Following transport into skeletal muscle, 2-DG is phosphorylated by hexokinase II to 2-deoxy-D-glucose-6-phosphate (2-DG-6P). As skeletal muscle lacks glucose-6-phophatase and GLUT4 cannot transport phosphorylated 2-DG, 2-DG-6P will be trapped within the muscle cell. In contrast to glucose-6-phosphate, 2-DG-6P is a very weak allosteric inhibitor of hexokinase II³⁰ which helps to maintain the transmembrane gradient for 2-DG. Thus, observations have shown that 2-DG uptake in incubated (rat) muscle remains linear until the intracellular 2-DG-6P concentration exceeds 30 mM, a concentration that lowers hexokinase II activity³⁰. Moreover, in incubated mouse skeletal muscle 2-DG uptake remains linear for ~30 min when temperatures of the incubation buffers are 37°C or less³¹. This suggests that 2-DG can be used to measure glucose transport capacity rather than glucose uptake in incubated muscle except for situations where 2-DG-6P concentrations become very high (e.g. observed during incubations >2 hours with 1 mM 2-DG and a maximal insulin concentration^29,30). The idea that 2-DG uptake likely reflects glucose transport capacity is also supported by findings showing that maximal insulin-stimulated 2-DG uptake is similar in incubated muscle from wild-type mice and mice that overexpress hexokinase II³². A potential concern when using 2-DG for muscle glucose transport measurements during contraction is an increase in the intracellular glucose-6-phosphate concentration due to an elevated rate of glycogenolysis. However, since a linear increase in (rat) muscle 2-DG uptake is observed during contraction over time²⁹, this indicates that the accumulation of glucose-6-phosphate from the breakdown of glycogen during contraction does not interfere with hexokinase II activity and thus 2-DG uptake rates. Based on this, 2-DG seems well suited for measurements of glucose transport in isolated skeletal muscle during insulin and contraction considering the limitations of 3-MG.

Although it is generally assumed that 2-DG is not further metabolized following phosphorylation by hexokinase II, it has been reported that some 2-DG is directed towards and incorporated into muscle glycogen. Thus, during a 2 h normoglycemic hyperinsulinemic clamp in rats ~30% of the 2-DG taken up by skeletal muscle is incorporated into glycogen³³. Therefore, it could be reasoned that rates of insulin-stimulated 2-DG uptake in incubated skeletal muscle are underestimated if accumulation of 2-DG in glycogen is neglected. We have determined that the herein described protocol on how to prepare muscle lysates (supernatant) for subsequent analyses of 2-DG uptake rates in prior incubated skeletal muscle is not affected by the potential incorporation of 2-DG into glycogen. It could be argued that glycogen accumulates in the pellet when centrifuging whole muscle homogenate to generate lysate for 2-DG uptake measurements. However, when comparing the levels of radioactivity in insulin-stimulated whole muscle homogenate vs. lysate we do not detect any significant difference (unpublished data). This suggests that incubating mouse muscle for 10 min in 1 mM of 2-DG does not cause a detectable accumulation of 2-DG in glycogen.

Determination of skeletal muscle 2-DG uptake without considering that 2-DG is distributed in both the extra- and intracellular space will lead to an overestimation of 2-DG uptake. To circumvent this, L-glucose should be used as an extracellular marker as this is not transported across the cell membrane but otherwise exhibits similar properties as D-glucose including mass, solubility, passive diffusion, binding, etc. Due to the excessive costs of manufacturing and thus purchasing L-glucose, mannitol is typically used as an extracellular marker since mannitol is not taken up by the muscle cell, is relatively inexpensive and is estimated to have a somewhat similar extracellular distribution volume as glucose and 2-DG³⁴.

Intrinsic cellular and molecular clocks seem to play an essential role for the regulation of whole-body metabolism and energy homeostasis³⁵. It has been observed that muscle-specific knockout of the core clock gene Bmal1 impairs insulin-stimulated glucose uptake in isolated mouse skeletal muscle³⁶. Furthermore, submaximal insulin-stimulated glucose uptake of isolated skeletal muscle exhibits circadian rhythm with the lowest and highest insulin response in the middle of the light and dark phase, respectively³⁷. Based on these findings, it is therefore important to incorporate time of animal sacrifice into the experimental design to increase the reproducibility of data.

A major disadvantage of the ex vivo method is the lack of capillary flow in the isolated muscle. This means that delivery and removal of various substrates fully depend on simple diffusion between the muscle fibers and the surrounding environment. Consequently, validation of the metabolic viability of isolated muscle incubated ex vivo has been focused on diffusion limitations of oxygen to superficial as well as deep muscle fibers. Thus, incubated muscle, particularly highly metabolic mouse muscle, tends to develop hypoxic cores in which breakdown of glycogen occurs^16,17,38. In a detailed review by Bonen and colleagues¹⁵, it was suggested that hypoxic cores of incubated muscle likely develop when incubating muscle too thick to be properly oxygenated especially when incubating at temperatures of ≥ 37 °C. This led to the recommendation that only thin and cylindrical mouse muscles, such as soleus and EDL, should be used for incubations at 25-30 °C to avoid development of hypoxic cores. This indicates that thickness and geometry rather than mass are important factors to consider when incubating mouse skeletal muscles. In addition, it was recommended that incubation temperature, muscle thickness as well as ATP, phosphocreatine, and glycogen content and/or release of lactate should be measured and reported routinely to evaluate the viability of the incubated muscle¹⁵. To our knowledge, such complete analyses of incubated muscle have mainly been reported for rat muscle^39,40,41,42 and only to a limited extent reported for mouse muscle^16,17. To increase insight into various metabolic markers of viability in incubated mouse skeletal muscle, we assessed possible changes in intracellular content of lactate, creatine, phosphocreatine, adenosine nucleotides, glycogen, and AMPK signaling in soleus and EDL muscle following 1 hour of incubation in glucose- or pyruvate-supplemented KRH buffer. Similar to previous findings in incubated mouse soleus and EDL muscle¹⁷, we observed that glycogen levels decreased when muscles were incubated in glucose-free media. This indicates that the absence of glucose during incubation promotes glycogen breakdown and the subsequent entry of glucose-6-phosphate into glycolysis that may act to secure ATP production in particular if oxygen supply is inadequate. Furthermore, we found an overall reduction in the ATP and ADP nucleotide pools in incubated soleus and EDL muscle. This may imply that oxygen supply is not entirely sufficient to meet the demand of incubated mouse muscle both in the presence and absence of glucose. Since the oxidative soleus muscle relies more on oxygen for ATP production compared to the glycolytic EDL muscle, this would suggest that the soleus muscle is affected to a larger extent by the incubation procedure. In agreement, we found that the intracellular levels of IMP as well as AMPK signaling were markedly increased in soleus compared to EDL muscle when incubated in the absence of glucose. This signifies that the soleus muscle exhibits a higher degree of metabolic stress during incubation, which must be taken into consideration when evaluating data from the ex vivo mouse muscle model.

Cultured muscle cells including immortalized L6 and C2C12 as well as primary human myotubes are commonly used as a surrogate for mature skeletal muscle to study the effects of various genetic and pharmacological manipulations on insulin-stimulated muscle glucose uptake. However, in several ways cultured muscle cells do not resemble mature skeletal muscle and when comparing the two model systems numerous differences become apparent. These include differences in protein expression, dimensional structure, surrounding environment, proliferation and differentiation state, fiber type composition, metabolic processes and functional properties⁴³ all of which may affect how the muscle cell regulates glucose uptake in response to various stimuli. Typically, greater relative effects of insulin on glucose uptake rates are observed in mature skeletal muscle compared to cultured muscle cells⁴⁴ which may indicate that cultured muscle cells to some extent lack the machinery responsible for regulating glucose uptake rates, including a high level of expression of the insulin-sensitive glucose transporter GLUT4⁴³. Considering the limitations and caveats associated with the use of either cultured muscle cells and isolated skeletal muscle, it is therefore likely advantageous to take on a combined approach when studying various muscle metabolic processes such as glucose uptake.

Soleus and EDL muscles are typically regarded as representative of slow- and fast-twitch muscles, respectively. Therefore, these muscle types are ideal for mechanical studies seeking to investigate interventions that affect muscle force and fatigue development. Moreover, soleus muscle is typically reported to have enhanced insulin sensitivity and responsiveness compared to EDL muscle and thus, interventions that target muscle insulin sensitivity may affect soleus and EDL differently. In addition, the relative distribution of the different AMPK complexes differs between the soleus and EDL muscle that seems to affect the ability of AMPK activating compounds to increase muscle glucose uptake. Thus, the greatest insight into the regulation of muscle glucose uptake is likely achieved if both the soleus and EDL muscles are used during experimentation with the ex vivo incubation model.

The herein described method relates glucose uptake to the amount of total protein abundance determined in each muscle sample after homogenization. It is our experience that variation decreases when relating glucose uptake per amount of muscle protein instead of muscle weight (unpublished data). Furthermore, homogenization of muscle samples in a buffer used for various biochemical assays makes it possible to determine both glucose uptake, myocellular signaling and enzyme activities in the same sample preparation^45,46. This will often decrease the amount of mice used for a study. Nevertheless, glucose uptake can easily be determined in skeletal muscle samples that are dissolved by heating in sodium hydroxide (NaOH) followed by neutralization with hydrogen chloride²⁰. Since treatment of muscle with NaOH interferes with measurements of total muscle protein concentration, glucose transport measured by this procedure must be related to muscle weight.

Based on various optimizations, our glucose uptake incubation buffer contains a specific activity of 0.028 MBq/mL of [³H]2-deoxy-D-glucose and 0.0083 MBq/mL of [¹⁴C]Mannitol. On one hand, this lowers the amount of lysate (150 out of 400 µL) needed in order to get sufficient and reliable radioactivity measurements. On the other hand, it increases the amount of radioactive [³H]2-deoxy-D-glucose and [¹⁴C]mannitol needed for each experiment, which increases experimental costs. Thus, for any experimental setup it is possible to regulate the amount of radioactivity used in the glucose uptake incubation media to fit specific requirements. However, care must be taken not to decrease amount of radioactivity in the incubation buffer to an extent that makes radioactivity measurements unreliable. This is ensured by keeping radioactivity in samples higher than the specified detection limits of the liquid scintillation counting machinery used.

Materials

Name	Company	Catalog Number	Comments
[14C]D-mannitol	American Radiolabeled Chemicals, Inc.	ARC 0127
[3H]2-deoxy-D-glucose	American Radiolabeled Chemicals, Inc.	ART 0103A
2-Deoxy-D-glucose	Sigma	D8375
4-0 USP non-sterile surgical nylon suture	Harvard Apparatus	51-7698
Streptavidin/HRP (Conjugate)	DAKO	P0397	Used to detect ACC protein
Akt2 antibody	Cell Signaling	3063
AMPKα2 antibody	Santa Cruz	SC-19131
aprotinin	Sigma	A1153
benzamidine	Sigma	B6505
Bovine serum albumin (BSA)	Sigma	A7030
CaCl2	Merck	1020831000
Calibration kit (force)	Danish Myo Technology A/S	300041
Chemiluminescence	Millipore	WBLUF0500
D-Glucose	Merck	1084180100
D-Mannitol	Sigma	M4125
Data collection program	National Instruments	LabVIEW software version 7.1
Dialysis tubing	Visking	DTV.12000.09 Size No.9
Digital imaging system	BioRad	ChemiDoc MP
EDTA	Sigma EDS	E9884
EGTA	Sigma	E4378
Electrical Pulse Stimulator	Digitimer	D330 MultiStim System
Glycerol	Sigma	G7757
HEPES	Sigma	H7637
IGEPAL CA-630	Sigma	I8896
Insulin	Novo Nordisk	Actrapid, 100 IE/mL
KCl	Merck	1049361000
KH2PO4	Merck	104873025
leupeptin	Sigma	L2884
MgSO4	Merck	1058860500
Muscle Strip Myograph System	Danish Myo Technology A/S	Model 820MS
Na-Orthovanadate	Sigma	S6508
Na-Pyrophosphate	Sigma	221368
Na-Pyruvate	Sigma	P2256
NaCl	Merck	106041000
NaF	Sigma	S1504
NaHCO3	VWR	27778260
pACC Ser212 antibody	Cell Signaling	3661
pAkt Thr308 antibody	Cell Signaling	9275
pAMPK Thr172 antibody	Cell Signaling	2531
phenylmethylsulfonylfluoride	Sigma	P7626
Platinum electrodes	Danish Myo Technology A/S	300145
pTBC1D4 Ser588 antibody	Cell Signaling	8730
Scintillation counter	Perkin Elmer	Tri-Carb-2910TR
Scintillation fluid	Perkin Elmer	6013329
Statistical analyses software	Systat	SigmaPlot version 14
TBC1D4 antibody	Abcam	ab189890
TissueLyser II	Qiagen	85300
Ultrapure water	Merck	Milli-Q Reference A+ System
β-glycerophosphate	Sigma	G9422